GSM6321096: D14-M1; Mus musculus; RNA-Seq
Enrichment for mesenchymal cells was carried out by negative selections using Magnetc-activated cell sorting (MACS). Cells were aliquoted in PBS supplemented with 0.04% of bovine serum albumin at a final concentration of 100 cells/µl. Using the microfluidic device, single cells (100/µl) were co-encapsulated in droplets with barcoded beads (120/µl, purchased from ChemGenes Corporation, Wilmington, MA) at rates of 4000 µl/hr. Droplet emulsions were collected for 10-20 min/each prior to droplet breakage by perfluorooctanol (Sigma-Aldrich). After breakage, beads were harvested and the hybridized mRNA transcripts reverse transcribed (Maxima RT, Thermo Fisher). Unused primers were removed by the addition of exonuclease I (New England Biolabs), following which, beads were washed, counted, and aliquoted for pre-amplification (2000 beads/reaction, equals ca. 100 cells/reaction) with 12 PCR cycles (Smart PCR primer: AAGCAGTGGTATCAACGCAGAGT (100 μM), 2x KAPA HiFi Hotstart Ready-mix (KAPA Biosystems), cycle conditions: 3 min 95°C, 4 cycles of 20s 98°C, 45s 65°C, 3 min 72°C, followed by 8 cycles of 20s 98°C, 20s 67°C, 3 min 72°C, then 5 min at 72°C). PCR products of each sample were pooled and purified twice by 0.6x clean-up beads (CleanNA), following the manufacturer's instructions. Prior to tagmentation, complementary DNA (cDNA) samples were loaded on a DNA High Sensitivity Chip on the 2100 Bioanalyzer (Agilent) to ensure transcript integrity, purity, and amount. For each sample, 1 ng of pre-amplified cDNA from an estimated 1000 cells was tagmented by Nextera XT (Illumina) with a custom P5-primer (Integrated DNA Technologies). Single-cell libraries were sequenced in a 100 bp paired-end run on the Illumina HiSeq4000 using 0.2 nM denatured sample and 5% PhiX spike-in. For priming of read 1, 0.5 μM Read1CustSeqB was used (primer sequence: GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC).